bmp2  (R&D Systems)

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Over-expressed Phospholamban in Cancer-associated Fibroblasts Is a Driver for CMS4 Subtype Colorectal Cancer Formation

doi: 10.1016/j.jcmgh.2025.101524

Figure Lengend Snippet: ( A ) qPCR analysis of angiogenesis marker genes mRNA levels in HUVEC cells qPCR analysis of angiogenesis marker genes mRNA levels in HUVEC cells incubated with CM from NFs transfected with empty vector, PLN overexpression (OE) and with/without anti-GREM1 neutralizing antibody (Ginisortamab, 10 μg/mL) for 48 hours. ( B ) qPCR assay for EMT-related genes of HCT116 and HT29 cells after BMP2, BMP4, BMP7 overexpressed or 20 ng/mL rBMP2, rBMP4, rBMP7 treatments with CAF CM incubation. ( C–D ) Representative photos and quantification of Transwell assay for HCT116 and HT29 cells after BMP2 overexpressed or 20 ng/mL rBMP2 treatment with CAF CM incubation. ( E–F ) Representative photos and quantification of wound healing assay for HCT116 and HT29 cells after BMP2 overexpressed or 20 ng/mL rBMP2 treatment with CAF CM incubation. ( G–H ) representative images of Transwell assay and quantification for cell invasion rates in empty vector or BMP2 OE transfected CT26, or wildtype CT26 treated with rBMP2 (20 ng/mL), all under incubation with mouse CAFs CM for 48 hours. ( I–K ) qPCR assay for angiogenesis-related genes of HUVEC cells after BMP2, BMP4, BMP7 overexpressed or 20 ng/mL rBMP2, rBMP4, rBMP7 treatments with CAF CM incubation. ( L–O ) qPCR assay for angiogenesis-related genes, as well as representative photos and quantification of tube formation assay in HUVEC cells after VEGFR2 knockdown with CAF CM incubation. ( P–R ) Representative images of tube formation assay ( P ), quantification of branching points ( Q ), and tube length ( R ) in C166 cells transfected with shCtrl, shVEGFR2 1#, or shVEGFR2 2# and incubated with mouse CAFs CM for 48 hours. ( S ) relative mRNA levels of PLN in human NFs after incubation with CM from NCM460 (normal colon epithelial cells)、primary CRC cancer cells (prCRC), and metastasis CRC cancer cells (mCRC) for 48 hours, as quantified by qPCR. ( T ) Representative Western blotting bands and quantification of PLN protein levels in NFs treated with CM from NCM460, prCRC, and mCRC. All experiments have been repeated at least 3 times independently. n.s., no significant differences; ∗∗ P < .01.

Article Snippet: Recombinant human BMP2 , Proteintech , HZ-1128.

Techniques: Marker, Incubation, Transfection, Plasmid Preparation, Over Expression, Transwell Assay, Wound Healing Assay, Tube Formation Assay, Knockdown, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Over-expressed Phospholamban in Cancer-associated Fibroblasts Is a Driver for CMS4 Subtype Colorectal Cancer Formation

doi: 10.1016/j.jcmgh.2025.101524

Figure Lengend Snippet: ( A–I ) qPCR analysis of efficiency BMP2, BMP4, and BMP7 overexpression in HCT116, HT29 and HUVEC cells. ( J ) qPCR analysis of shRNA interference efficiency targeting VEGFR2 in HUVEC cells. ( K–N ) qPCR analysis of shRNA interference efficiency targeting ITGB1, MET, EFBN1, and CSF1R in human NFs. ( O–S ) qPCR analysis of shRNA interference efficiency targeting COL3A1, VTN, HGF, EPHA6, and CSF1 in mCRC cells. All experiments have been repeated at least 3 times independently. n.s., no significant differences; ∗∗ P < .01.

Article Snippet: Recombinant human BMP2 , Proteintech , HZ-1128.

Techniques: Over Expression, shRNA

Journal: PLOS ONE

Article Title: IRX-related homeobox gene MKX is a novel oncogene in acute myeloid leukemia

doi: 10.1371/journal.pone.0315196

Figure Lengend Snippet: (A) Genomic copy number analysis of OCI-AML3 showing the complete chromosome 10 (above), and the chromosomal region 10p12.1-p12.3 highlighting MLLT10 and MKX (below). (B) Data obtained from the UCSC genome browser indicating a CpG-island at MKX (left). RQ-PCR analysis of MKX and NANOG in AML cell line HL-60 treated with DNA-methyltransferase inhibitor AZA (right). The expression level of HL-60 control was set as 1 while the remaining samples are related to that value. (C) RQ-PCR analysis of IRX3 and MKX in OCI-AML3 treated with BMP2 (left) and dorsomorphin (middle). RQ-PCR analysis of SMAD4 , IRX3 and MKX in OCI-AML3 treated for siRNA-mediated knockdown (right). (D) RQ-PCR analysis of IRX3 and MKX (left), and IRX5 and MKX (right) in OCI-AML3 treated for siRNA-mediated knockdown. (E) RQ-PCR analysis of JUNB and MKX in OCI-AML3 treated for siRNA-mediated knockdown. The expression level of OCI-AML3 siCTR was set as 1 while the remaining samples are respectively related to that value. Statistical significance was assessed by Student´s T-Test (two-tailed) and the resultant p-values indicated by asterisks (* p<0.05, ** p<0.01, *** p<0.001, n.s. not significant).

Article Snippet: Additional cell treatments were performed using 20 ng/ml recombinant BMP2 or TNFSF11 (R & D Systems, Wiesbaden, Germany), 100 μM azacytidine (AZA), 10 μM dorsomorphin, 14 μM NFkB-inhibitor, and 100 μM etoposide (Sigma-Aldrich, Taufkirchen, Germany) for 20 h. For functional testing, treated cells were analyzed using the IncuCyte S3 Live-Cell Imaging Analysis System and the Cell-by-Cell analysis software (Sartorius, Göttingen, Germany).

Techniques: Expressing, Control, Knockdown, Two Tailed Test

Journal: bioRxiv

Article Title: Mature tuft cell phenotypes are sequentially expressed along the intestinal crypt-villus axis following cytokine-induced tuft cell hyperplasia

doi: 10.1101/2024.11.28.625899

Figure Lengend Snippet: (a) Culture setup to test tuft cell differentiation. Tuft-type reporter organoids were pretreated with IL-4 and IL13 on the 3 rd day after seeding in ENR (EGF, R-spondin and Noggin) medium. On the 4 th day, various factors were added to, or depleted from the medium. Frequency of tuft-type induction was tested at day 7 with flow cytometry. (b) Left, percentage of ChAT-eGFP + cells in organoids treated with IL-4 and IL-13 in presence or absence of crypt or villus/autocrine-inspired signaling factors. Right, fold change induction of ChAT-eGFP + cells after addition of single villus/autocrine-inspired factors to cytokine-pretreated organoids without ENR. Data are represented as mean ± SD (ANOVA p < 0.0001, *** adjusted p-value < 0.001, **** adjusted p-value < 0.0001, Tukey HSD test). (c) Representative flow cytometry analysis of fluorescent population frequencies in indicated tuft-type reporter organoids treated with ENR and cytokines (crypt+IL4+IL13) or villus-inspired medium (no ENR; with IL-25, WNT5a, NRG1, BMP2, BMP4 and acetylcholine chloride) after IL-4 and IL-13 pretreatment. (d) Microscopic stills from live imaging experiments of organoids in villus-inspired medium (no ENR; with IL-25, WNT5a, NRG1, BMP2, BMP4 and acetylcholine chloride) after IL-4 and IL-13 pretreatment. Left: confocal brightfield image of ChAT BAC -eGFP; Nrep P2A-mScarlet-I organoid at the start of imaging. Right: stills of fluorescence channels (mSarlet-I: red, eGFP: green) from timepoints preceding and following co-expression of fluorescent markers. Cell that shows co-expression is indicated with a white arrowhead throughout imaging timepoints. (e) Average mScarlet-I signal over time in Nrep P2A-mScarlet-I organoids in medium with ENR and cytokines (top; crypt) or villus-inspired medium (no ENR; with IL-25, WNT5a, NRG1, BMP2, BMP4 and acetylcholine chloride) after IL-4 and IL-13 pretreatment (bottom; villus). Shading in plot represents SEM. Number of cells comprising each graph are indicated. (f) Barplot showing fraction of mScarlet-I + cells in Nrep P2A-mScarlet-I organoids with stable/rising fluorescence signal or declining fluorescence signal in crypt or villus medium with cytokines (IL4+IL13). Related to e. Number of cells comprising each graph are indicated. (g) Transcriptomic analysis of single cells in organoids upon induction with cytokines in ENR or villus-inspired culture conditions. Left, UMAP of organoid cells being enriched for tuft cells by FACS. Cell types are annotated by color. EE: enteroendocrine cells. Right, same UMAP but cells are colored by medium condition. (h) Violin plots depicting distribution of relative levels of tuft signature scores in tuft-1 cells (left) or tuft-2 cells (right) origating from different culture conditions (ns not significant, ** p-value < 0.01, *** p-value < 0.001, Wilcoxon rank sum test). (i) RNA velocity-based trajectory inference superimposed on UMAP of predicts unidirectional differentiation from tuft-1 to tuft-2 transcriptomic states.

Article Snippet: Thereafter, tuft cell maturation was facilitated by removal of EGF, Noggin and R-spondin from the medium and addition of one of the following or a combination thereof on the 4 th day after passaging: recombinant murine IL-25 (20 ng/ml, Immunotools), recombinant human BMP2 (20 ng/ml, Immunotools), recombinant human BMP4 (20 ng/ml, Immunotools), recombinant human NRG1 (20 ng/ml, R&D systems), recombinant murine WNT5a (20 ng/ml, Biotechne) and acetylcholine chloride (100 μM, Sigma Aldrich).

Techniques: Cell Differentiation, Flow Cytometry, Imaging, Fluorescence, Expressing

Journal: Bioactive Materials

Article Title: Nanoclay gels attenuate BMP2-associated inflammation and promote chondrogenesis to enhance BMP2-spinal fusion

doi: 10.1016/j.bioactmat.2024.10.027

Figure Lengend Snippet: Overview of the subfascial implantation mouse model. Following implantation of test materials beneath the fascia, the following evaluations were performed at the indicated time points: release kinetics of BMP2 and inflammatory response (on day 7), chondrogenesis (on days 7, 10, 14, and 21), osteoclastogenesis (on days 7, 10, 14, 21 and 42), and osteogenesis (on day 42).

Article Snippet: Escherichia coli -derived recombinant human BMP2 used in this study was kindly provided by CGBio (Seoul, Korea), and was adjusted to 1 μg/μL.

Techniques:

Journal: Bioactive Materials

Article Title: Nanoclay gels attenuate BMP2-associated inflammation and promote chondrogenesis to enhance BMP2-spinal fusion

doi: 10.1016/j.bioactmat.2024.10.027

Figure Lengend Snippet: Retention kinetics of fluorescently labeled BMP2. (A) In vitro fluorescence image of CS and NC containing fluorescently labeled 0.1, 0.5, and 1 μg of BMP2 at the bottom of a 24-well plate. In vitro radiant efficiency up to 7 days; (B) 0.1 μg of BMP2; (C) 0.5 μg of BMP2; (D) 1 μg of BMP2 . Retention kinetics of NC and CS containing 1 μg BMP2 up to 24 h and up to 7 days (E, F). (G) In vivo fluorescence image of CS and NC containing fluorescently labeled 1 and 2 μg of BMP2 in a mouse subfascial implantation model. In vivo radiant efficiency up to 7 days: (H) 1 μg of BMP2; (K) 2 μg of BMP2. Retention kinetics of NC and CS containing 1 μg of BMP2 up to 24 h and up to 7 days (I, J). Retention kinetics of NC and CS containing 2 μg of BMP2 up to 24 h and up to 7 days (L, M). Data represents the mean ± SD (n = 4 in each group).

Article Snippet: Escherichia coli -derived recombinant human BMP2 used in this study was kindly provided by CGBio (Seoul, Korea), and was adjusted to 1 μg/μL.

Techniques: Labeling, In Vitro, Fluorescence, In Vivo

Journal: Bioactive Materials

Article Title: Nanoclay gels attenuate BMP2-associated inflammation and promote chondrogenesis to enhance BMP2-spinal fusion

doi: 10.1016/j.bioactmat.2024.10.027

Figure Lengend Snippet: Evaluation of bone and cartilage formation in the subfascial implantation model. (A) Representative micro-CT images in the CS and in NC groups on day 21 and day 42. Scale bars, 1 mm. (B) Bone mineral density (BMD) of the implanted materials: CS + PBS (n = 6), NC + PBS (n = 6), CS + BMP2 (n = 6), and NC + BMP2 (n = 6). (C) Representative H&E images on day 21 and 42. The NC group showed extensive amounts of bone formation within NC. Scale bars, 1 mm (upper panels), 500 μm (lower panels). (D) Amounts of newly formed bone within the carriers and thickness of the outer cortical shell in the CS and NC groups (n = 6 each). (E) Time-dependent cartilage formation in CS and NC containing 1 μg of BMP2 as evaluated by safranin-O staining. Scale bars, 1 mm (upper panels), 200 μm (lower panels). (F) Quantification of the safranin-O-stained area on day 21 in the CS and NC groups (n = 5 each). Scale bars, 1 mm. (G) Quantitative analysis of sGAG in the CS and NC groups (n = 4 each). Data represent mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001 as determined via an unpaired t -test (D, F) or via two-way ANOVA, followed by Bonferroni tests (B, G).

Article Snippet: Escherichia coli -derived recombinant human BMP2 used in this study was kindly provided by CGBio (Seoul, Korea), and was adjusted to 1 μg/μL.

Techniques: Micro-CT, Staining

Journal: Bioactive Materials

Article Title: Nanoclay gels attenuate BMP2-associated inflammation and promote chondrogenesis to enhance BMP2-spinal fusion

doi: 10.1016/j.bioactmat.2024.10.027

Figure Lengend Snippet: Effect of NC on the inflammatory response in the subfascial implantation model. (A) Representative images of H&E-staining sections of subfascial implanted CS and NC containing different concentrations of BMP2 on day 7. BMP2 dose-dependent inflammatory cell infiltration was not observed in the NC group, in contrast to the CS group. Scale bar, upper row: 1 mm, lower row: 300 μm. (B) Quantitative analysis of the area and thickness of the inflammatory lesion in the CS and NC groups (n = 3 each). (C) Immunostaining for CD68, neutrophils, CD206 and TRAP staining of the inflammatory zone around CS and NC containing 5 and 10 μg of BMP2. Scale bars, 100 μm. (D) Quantification of CD68-positive monocytes, neutrophils, TRAP-positive cells, and CD206-positive multinucleated giant cells (MNGCs) (red arrow). Data represent the mean ± SD. ∗ p < 0.05, ∗∗∗∗ p < 0.0001, as determined via two-way ANOVA, followed by Bonferroni tests.

Article Snippet: Escherichia coli -derived recombinant human BMP2 used in this study was kindly provided by CGBio (Seoul, Korea), and was adjusted to 1 μg/μL.

Techniques: Staining, Immunostaining

Journal: Bioactive Materials

Article Title: Nanoclay gels attenuate BMP2-associated inflammation and promote chondrogenesis to enhance BMP2-spinal fusion

doi: 10.1016/j.bioactmat.2024.10.027

Figure Lengend Snippet: Assessment of inflammatory responses as assessed by immunostaining. (A) Representative TNF-α immunostaining images for both CS and NC groups. Scale bars, 1 mm (upper panels), 100 μm (lower panels). In the CS group, inflammatory cells were observed infiltrated on the outside (black arrows) rather than inside (blue arrows) of the implant, whereas in the NC group, inflammatory cells could barely be detected outside the NC (black arrow). (B) Quantification of TNF-α-positive cell number (n = 3 each). (C) Representative Sox9 immunostaining images in the inflammatory area around CS and NC containing 5 μg of BMP2. (D) Quantification of SOX9-positive cells inside and outside of the implanted materials (n = 4 each). Scale bars, 100 μm. Data represent the mean ± SD. ∗ p < 0.05, ∗∗∗∗ p < 0.0001, as determined via two-way ANOVA, followed by Bonferroni tests.

Article Snippet: Escherichia coli -derived recombinant human BMP2 used in this study was kindly provided by CGBio (Seoul, Korea), and was adjusted to 1 μg/μL.

Techniques: Immunostaining